Human serum cholinesterase is of pharmacogenetic interest since deficiencies of variant forms of this enzyme cause an exaggerated response to the muscle-relaxant drug, succinylcholine. Our laboratory has studied this enzyme for the past 25 years. We have determined the complete amino acid sequence of the enzyme, cloned the gene from a human brain cDNA library, and sequenced the coding region of this protein. During the last two years, we found that the structural basis for the most common qualitative variant, the atypical (dibucaine-resistant) variant, is a point mutation at nucleotide base 209 (GAT->GGT); this changes the amino acid at codon 70 from Glu->Gly. This same mutation has been found in every individual with the atypical phenotype. We used the polymerase chain reaction technique to amplify a selected segment of WBC DNA which included the mutation region, followed by direct sequencing. During the next three years we will identify the structural basis (DNA alterations) responsible for a number of the other qualitative variants, such as the fluoride- resistant variant, and several quantitative variants, such as the K-variant (reduction of 1/3); the J-variant (reduction of 2/3), and the H-variant (reduction of 90%) as well as some of the "silent" variant which accounted for by a frame-shift mutation at nucleotide 351 (GGT->GGAG). These analyses will provide a DNA structural basis for classifying cholinesterase genotypes, and improve our ability to identify those individuals who would be sensitive to succinylcholine, if given the drug. Simple non-radioactive, allele-specific biotinylated probes are being developed to easily and quickly identify the variant genotypes, after PCR amplification of the respective DNA segments from white blood cell DNA. These studies will identify a number of amino acids that have a critical role in the catalytic activity of the enzyme, and some that particularly influence the half-life or stability of the enzyme.